• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.8-15
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• 2021

More and more available fungal genome sequence data reveal a large amount of secondary metabolite (SM) biosynthetic 'dark matter' to be discovered. Heterogeneous expression is one of the most effective approaches to exploit these novel natural products, but it is limited by having to clone entire biosynthetic gene clusters (BGCs) without errors. So far, few effective technologies have been developed to manipulate the specific large DNA fragments in filamentous fungi. Here, we developed a fungal BGC-capturing system based on CRISPR/Cas9 cleavage in vitro. In our system, Cas9 protein was purified and CRISPR guide sequences in combination with in vivo yeast assembly were rationally designed. Using targeted cleavages of plasmid DNAs with linear (8.5 kb) or circular (8.5 kb and 28 kb) states, we were able to cleave the plasmids precisely, demonstrating the high efficiency of this system. Furthermore, we successfully captured the entire Nrc gene cluster from the genomic DNA of Neosartorya fischeri. Our results provide an easy and efficient approach to manipulate fungal genomic DNA based on the in vitro application of Cas9 endonuclease. Our methodology will lay a foundation for capturing entire groups of BGCs in filamentous fungi and accelerate fungal SMs mining.

• The Korean Society of Law and Medicine
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• v.23 no.1
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• pp.121-148
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• 2022

CRISPR-Cas9 is one of the gene-editing technologies that infinite potential. It may provide human beings with many benefits or cause unanticipated challenges. The governance as standards setting or regulation of gene-editing technologies can contribute to keeping a balance between scientific value and ethical commitments. Guaranteeing public participation provides an additional opportunity to think about ethical and moral considerations: For whose benefit the internationally discussed governance of gene-editing technologies is directed at? There is a doubt regarding whether the governance justifies scientific researchers' gene-editing research. Suppose that governance promotes the advancement of CRISPR-Cas9, it should also encourage greater research responsibility. If not, there may be tragedies brought about by the misconduct of researchers. Thus, the essential matter on the governance for the research of CRISPR-Cas9 is the researchers' responsibility.

• Molecules and Cells
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• v.38 no.6
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• pp.475-481
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• 2015

Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations.

• Journal of Plant Biotechnology
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• v.48 no.1
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• pp.34-43
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• 2021

Targeted genome editing using the CRISPR/Cas9 system is a ground-breaking technology that is being widely used to produce plants with useful traits. However, for woody plants, only a few successful attempts have been reported. These successes have used Agrobacterium-mediated transformation, which has been reported to be very efficient at producing genetically modified trees. Nonetheless, there are unresolved problems with plasmid sequences that remain in the plant genome. In this study, we demonstrated a DNA-free genome editing technique in which purified CRISPR/Cas9 ribonucleoproteins (RNPs) are delivered directly to the protoplasts of a hybrid poplar (Populus alba × Populus glandulosa). We designed three single-guide RNAs (sgRNAs) to target the stress-associated protein 1 gene (PagSAP1) in the hybrid poplar. Deep sequencing results showed that pre-assembled RNPs had a more efficient target mutagenesis insertion and deletion (indel) frequency than did non-assembled RNPs. Moreover, the RNP of sgRNA3 had a significantly higher editing efficacy than those of sgRNA1 and sgRNA2. Our results suggest that the CRISPR/Cas9 ribonucleoprotein-mediated transfection approach is useful for the production of transgene-free genome-edited tree plants.

• International Journal of Industrial Entomology and Biomaterials
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• v.39 no.1
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• pp.14-21
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• 2019

Genome editing by CRISPR/Cas9, a third-generation gene scissor in molecular breeding at the genome level, is attracting much attention as one of the breeding techniques of the future. In this study, genetic and phenotypic analysis was used to examine the responsiveness of the Bakokjam variety of the silkworm Bombyx mori to molecular breeding using CRISPR/Cas9 in editing the white egg 2 (w-2) gene. The nucleotide sequence of the w-2 gene was analyzed and three different guide RNAs (gRNA) were prepared. The synthesized gRNA was combined with Cas9 protein and then analyzed by T7 endonuclease I after introduction into the Bm-N silkworm cell line. To edit the silkworm gene, W1N and W2P gRNA and Cas9 complexes were microinjected into silkworm embryos. Based on the results of microinjection, the hatching rate was 16-24% and the incidence of mutation was 33-37%. The gene mutation was verified in the heterozygous F1 generation, but no phenotypic change was observed. In F2 homozygotes generated by F1 self-crosses, a mutant phenotype was observed. These results suggest that silkworm molecular breeding using the CRISPR/Cas9 system is possible and will be a very effective way to shorten the time required than the traditional breeding process.

• Proceedings of the Korean Society of Crop Science Conference
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• 2022.10a
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• pp.285-285
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• 2022

Recent unpredictable climate change is a major cause of rice yield loss. In particular, methane is a key factor in global warming. Therefore rice breeders are trying to breed the reducing-methane gas emission rice using the crossbreeding method. However, the traditional crossbreeding method takes 8 to 10 years to breed a cultivar, and the anther culture method developed to shorten the breeding cycle also takes 6 to 7 years. On the other hand, CRISPR/Cas9 accurately edits the target trait and can rapidly breed rice cultivars by editing the target trait as a homozygous in 2-3 years. In addition, exogenous genetic elements such as Cas9 can be isolated from the G₁ generation. Therefore, the flowering time was regulated by applying CRISPR/Cas9 technology, and OsCKq1 genome-editing (OsCKq1-G) rice with early flowered and high yield was bred in the field. Genome-editing of OsCKq1 applied CRISPR/Cas9 technology up-regulates the expression of the flowering promotion gene Ehd1 under long-day conditions induces early flowering and increases the yield by increasing the 1,000-grain weight. And as the generations advanced, each agricultural trait indicated a low coefficient of variation. As a result, indicated that OsCKq1 plays an important role in regulating the flowering time and is related to the trait determining yield. Therefore, OsCKq1-G can suggest a breeding strategy for the Net-Zero national policy for reducing-methane gas emission rice by shortening the breeding cycle with the early flowered, and high-yield rice. CRISPR/Cas9 technology is a rapid and accurate breeding technology for breeding rice cultivars with important characteristics.

• Microbiology and Biotechnology Letters
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• v.49 no.4
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• pp.534-542
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• 2021

Single-molecular guide RNA (sgRNA) plays a role in recognizing the DNA target sequence in CRISPR technology for genome editing and gene expression control. In this study, we systematically compared the length of the target recognition sequence in sgRNAs required for genome editing using Cas9-NG (an engineered Cas9 recognizing 5'-NG as PAM sequence) and gene expression control using deactivated Cas9-NG (dCas9-NG) by targeting the gal promoter in E. coli. In the case of genome editing, the truncation of three nucleotides in the target recognition sequence (TRS) of sgRNA was allowed. In gene expression regulation, we observed that target recognition and binding were possible even if eleven nucleotides were deleted from twenty nucleotides of the TRS. When 4 or more nucleotides are truncated in the TRS of the sgRNA, it is thought that the sgRNA/Cas9-NG complex can specifically bind to the target DNA sequence, but lacks endonuclease activity to perform genome editing. Our study will be helpful in the development of artificial transcription factors and various CRISPR technologies in the field of synthetic biology.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.4
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• pp.323-328
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• 2020

Direct injection of genome editing tools such as CRISPR/Cas9 system into developing embryos has been widely used to generate genetically engineered pigs. The approach allows us to produce pigs carrying targeted modifications at high efficiency without having to apply somatic cell nuclear transfer. However, the targeted modifications during embryogenesis often result in mosaicism, which causes issues in phenotyping founder animals and establishing a group of pigs carrying intended modifications. This study was aimed to establish a genomic PCR and sequencing system of a single blastomere in the four-cell embryos to detect potential mosaicism. We performed genomic PCR in four individual blastomeres from four-cell embryos. We successfully amplified target genomic region from single blastomeres of 4-cell stage embryo by PCR. Sanger sequencing of the PCR amplicons obtained from the blastomeres suggested that PCR-based genotyping of single blastomere was a feasible method to determine mutation type generated by genome editing technology such as CRISPR/Cas9 in early stage embryos. In conclusion, we successfully genotyped single blastomeres in a single 4-cell stage embryo to detect potential mosaicism in porcine embryos. Our approach offers a simple platform that can be used to screen the prevalence of mosaicism from designed CRISPR/Cas9 systems.

• Microbiology and Biotechnology Letters
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• v.49 no.2
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• pp.210-216
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• 2021

This study aimed to enhance ethanol productivity of Saccharomyces cerevisiae through genome editing using CRISPR/CAS9. To increase ethanol productivity, ACC1, ELO1, and OLE1 were overexpressed in S. cerevisiae using the CRISPR/CAS9 system. The strains overexpressing ACC1, ELO1, and OLE1 survived up to 24 h in YPD medium supplemented with 18% ethanol. Moreover, the ethanol yields in strains overexpressing ACC1 (428.18 mg ethanol/g glucose), ELO1 (416.15 mg ethanol/g glucose), and OLE1 (430.55 mg ethanol/g glucose) were higher than those in the control strains (400.26 mg ethanol/g glucose). In conclusion, the overexpression of these genes increased the viability of S. cerevisiae at high ethanol concentrations and the ethanol productivity without suppressing glucose consumption.

• Korean Journal of Environment and Ecology
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• v.35 no.5
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• pp.503-524
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• 2021

A largemouth bass (Micropterus salmoides) is an ecosystem disturbance fish species at the highest rank in the aquatic ecosystem, causing a serious imbalance in freshwater ecosystems. Although various attempts have been made to eradicate and control largemouth bass, no effective measures were found. Therefore, it is necessary to find an approach to maximize the effective population reduction based on the unique characteristics of largemouth bass. This study used the transcriptome analysis to derive 182,887 unigene contigs and select 12 types of final target sequences for applying the CRISPR/Cas9 system in the genes of IZUMO1 and Zona pellucida sperm-binding protein, which are proteins involved in sperm-egg recognition. After synthesizing 12 types of sgRNA capable of recognizing each target sequence, 12 types of Cas9-sgRNA ribonucleoprotein (RNP) complexes to be used in subsequent studies were prepared. This study searched the protein-coding gene of sperm-egg through the Next Generation Sequencing (NGS) and edited genes through the CRISPR/Cas9 system to induce infertile individuals that produced reproductive cells but could not form fertilized eggs. Through such a series of processes, it successfully established a composition development process for largemouth bass. It is judged that this study contributed to securing the valuable basic data for follow-up studies to verify its effect for the management of ecological disturbances without affecting the habitat of other endemic species in the same water system with the largemouth bass.